Abstract

Introduction: The major complications of stent implantation are restenosis and late stent thrombosis. PBMA polymers are used for stent coating because of their mechanical properties. We previously synthesized and characterized Dextrangraft-polybutylmethacrylate copolymer (Dex-PBMA) as a potential stent coating. In this study, we evaluated the haemocompatibility and biocompatibility properties of Dex-PBMA in vitro and in vivo. Methods: Here, we investigated: (1) the effectiveness of polymer coating under physiological conditions and its ability to release Tacrolimus®, (2) the capacity of Dex-PBMA to inhibit Staphylococcus aureus adhesion, (3) the thrombin generation and the human platelet adhesion in static and dynamic conditions, (4) the biocompatibility properties in vitro on human endothelial colony forming cells ( ECFC) and on mesenchymal stem cells (MSC) and in vivo in rat models, and (5) we implanted Dex-PBMA and Dex-PBMATAC coated stents in neointimal hyperplasia restenosis rabbit model. Results: Dex-PBMA coating efficiently prevented bacterial adhesion and release Tacrolimus®. Dex-PBMA exhibit haemocompatibility properties under flow and ECFC and MSC compatibility. In vivo, no pathological foreign body reaction was observed neither after intramuscular nor intravascular aortic implantation. After Dex-PBMA and Dex-PBMATAC coated stents 30 days implantation in a restenosis rabbit model, an endothelial cell coverage was observed and the lumen patency was preserved. Conclusion: Based on our findings, Dex-PBMA exhibited vascular compatibility and can potentially be used as a coating for metallic coronary stents.

The synthesis leads to a homogeneous white powder of dextran-graft-polybutylmethacrylate copolymer (Dex-PBMA ) soluble in a mixture of THF and water (92:8 v/v). The coatings of Dex-PBMA on a glass coverslip, CoCr disc and CoCr stent or moulding resulted in a transparent film, homogeneous and without cracks. Dex-PBMA deep-coating of stent covered each strut independently with very few webbing. The stent surface modifications were studied using SEM and contact-angle analysis. As shown , this coating withstands to the crimping on the balloon and the deployment. After one week of incubation at 37°C and under coronary-like flow conditions, Dex-PBMA coating remained unaltered and showed a uniform polymer surface, niether pealing nor delamination was observed.

As shown , Staphylococcus aureus was lower on Dex-PBMA than on CoCr (ratio 11, P < 0.005) and control collagen (ratio 4.5, P < 0.05). In contrast, more bacteria adhered on CoCr than on control (ratio 4.7, P < 0.005).

In static condition, platelet adhered less on Dex-PBMA () than on the positive control collagen (ratio 2.7, P < 0.0001) or on CoCr (ratio 1.39, P < 0,01). The SEM images showed less platelet activation on the copolymer than on the bare metal. Non-activated platelets are disk-shaped. Once activated, they first take a spherical shape as observed on Dex-PBMA disk () before spreading and giving out extensions such as filopodia that can be observed on CoCr surface. In dynamic conditions, no platelet adhered on Dex-PBMA (). Dex-PBMA coating does not promote platelet adhesion and activation in our experimental conditions.

ECFCs and MSCs adhered less on Dex-PBMA disc than on CoCr disc (). Non-significant differences were obtained for intermediate adhesion (20 min incubation time) whereas for focal adhesion (2 hours incubation time), 2.4 less ECFCs (, P < 0.0005) and 1.6 less MSCs (, P < 0.05) adhered on Dex-PBMA compared to CoCr discs. The same trends were observed for cell proliferation ( and ). The proliferation of ECFCs and MSCs was greater on CoCr disc than on Dex-PBMA . In both cellular type and surfaces, the proportion of cells compared to control coating of gelatin decrease after 4 days. Thus, CoCr or Dex-PBMA surfaces cause cells to grow more slowly than on control gelatin wells.

Dex-PBMA films were first implanted in rat abdominal wall to evaluate the tissue reactions after 7 and 30 days (). As shown on HE staining (), a tissue surrounded the film at day 7 with a thickness of 40 ± 12 µm that remain stable at day 30 (47 ± 12 µm). The low level of Naphthol AS-D Chloroacetate staining indicated that no neutrophil granulocytes were detected in the tissue (). The capsule is thus mainly composed by collagen without any evidence of inflammatory reaction or necrosis.

Dex-PBMA was then evaluated on a coated stent in rat abdominal aorta. Immediately after implantation, no acute thrombosis was observed. After 30 days, all Dex-PBMA or control CoCr stent implanted rats (n=6) survived. At day 30, angiographies confirmed that all arteries were patent. As shown in , no thrombus was found in Dex-PBMA or CoCr stented arteries. Nevertheless, a small intimal hyperplasia was observed both on Dex-PBMA and CoCr stents whereas no significant differences were noticed in the areas of intima (0.54 ± 0.007 mm2 vs. 0.50 ± 0.06 mm2) and media (0.40 ± 0.04 mm2 vs. 0.43 ± 0.02 mm2) on CoCr stent and Dex-PBMA stent (). No macrophages were emphasized in the arterial wall (CD 68 staining, ). The neointima was mainly composed of SMCs (α-actin staining). This SMC layer was cover by an endothelial layer (RECA staining) on Dex-PBMA as well on CoCr stents (). As observed in vitro, no platelets adhered on Dex-PBMA and we noticed the absence of thrombus formation.

Dex-PBMA or Dex-PBMA TAC stents were evaluated in a pathological model of neointimal hyperplasia. After 30 days the iliac arteries were patent. Hematoxylin-eosin staining indicated a neointima (). The measurements emphasized a higher ratio of intima and media areas on Dex-PBMA stent than on CoCr stent (2.39 vs 1.90). Nevertheless, with Dex-PBMA TAC this ratio decreased (1.60 vs 2.39). On the other hand, TNF-α blood concentration used as a biomarker between before and 30 days after implantation increased in CoCr stent implanted rabbit (+15.6 pg/mL), and remain stable for Dex-PBMA stent implanted rabbit (+0.53 pg/mL) and decreased for Dex-PBMA TAC stent implanted rabbit (-4.87 pg/mL) ().